The present invention relates to a device which is adaptable for use with a flow cytometer to decelerate electrically charged droplets formed by the flow cytometer, in order to minimize damage to the cells contained in the droplets when the droplets are collected in corresponding collection vessels. More particularly, the device includes a plurality of deceleration devices arranged in correspondence with the collection vessels into which charged droplets traveling along different paths in a flow cytometer are collected, with each deceleration device creating an electrostatic field which repels and thus slows the movement of the droplets so that the droplets enter the corresponding collection vessels at reduced speeds.
Flow cytometers for sorting and examining biological cells are well known in the art. Known flow cytometers are described, for example, in U.S. Pat. Nos. 4,347,935, 5,464,581, 5,483,469, 5,602,039 and 5,643,796, the entire contents of which are incorporated by reference herein. Another known flow cytometer is the FACS Vantage.TM. system manufactured by Becton Dickinson and Company, the assignee of the present invention. A flow cytometer typically includes a sample reservoir for receiving a biological sample, such as a blood sample. The sample contains cells that are to be analyzed and sorted by the flow cytometer.
Physical and fluorescent properties of particles hereinafter called cells can be measured as they intersect a laser beam. This is accomplished by transporting cells in a cell stream to a flow cell. Within the flow cell, a liquid sheath is formed around the cell stream to impart a uniformly velocity and to hydrodynamically focus the cells within the stream onto the center of a laser beam. The point of intersection or interrogation point can be inside or outside the flow cell. As a cell moves through the interrogation point, it causes the laser light to scatter and fluorescent molecules either within the cell or added to the cell becomes excited.
An appropriate detection system consisting of photomultipliers tubes, photodiodes or other devices for measuring light are focused onto the intersection point where the properties are measured. To sort cells by an electrostatic method, the desired cell must be contained within an electrically charged droplet. To produce the droplet, the flow cell is rapidly vibrated by an acoustical device. These droplets form after the cell stream exits the flow cell and at a distance from the interrogation point so that a time delay is required between the interrogation point and the actual break off point of the droplet. This time delay is determined by appropriately designed electronic circuits that are synchronized with the device that forms the droplets.
To charge the droplet, the cell stream passes by or through a charging power whose electrical potential relative to a charge generated in the sheath fluid can be rapidly changed. At the instant the desired cell is in the droplet just breaking away from the cell stream, the charging power is brought up to potential thereby causing the droplet to isolate the charge once broken off from the stream. The electrostatically charging device can cycle to appropriately charge each droplet as it is being broken off the cell stream.
Because the cell stream exits the flow cell in a substantially downward vertical direction, the droplets also propagate in that same direction after they are formed. To sort the charged droplet containing the desired cell, they are deflected from the trajectory of uncharged droplets as they pass through an electrostatic field formed by two deflection plates held constant at a high electrical potential difference. Positively charged droplets are attracted by the negative plate and repelled by the positive plate while negatively charged droplets are attracted to the positive plate and repelled by the negative plate. This causes their trajectory to become changed thereby sorting them from other cells. Because of their high velocity and because of the length of the plate, the cells clear the deflection field before striking the deflection plates. Accordingly, the droplets in the cells contained therein can be collected in their appropriate collecting vessels.
Although flow cytometers of the type described above are generally effective in sorting and analyzing cells, the speed at which these systems can operate is limited by the physical fragility of the cells. That is, to increase the rate at which the droplets are formed, analyzed and sorted, it is necessary to increase the pressure in the sheath fluid to thereby increase the flow rate of the fluid jet. Although some flow cytometers are capable of performing the required analyzing and sorting operations as the droplet flow rate is increased, the increased velocity of the droplets can result in the droplets striking the interior of the collection vessels with a force sufficient to damage or rupture the cells contained in the droplets. Typically, a sheath fluid pressure of greater than 40 psi will cause the droplets to be ejected from the nozzle at a speed great enough to cause damage to the cells when the droplets strike the interior of the collection vessels. By maintaining the pressure of the sheath fluid below these levels to avoid damage to the cells, it is not possible to sort cells at interrogation rates greater than about 2,000 cells per second.
Accordingly, a need exists for a flow cytometer which is capable of operating at an increased droplet flow rate while avoiding damage to the cells contained in the droplets.